Ultrasensitive Immunosensor Based on Langmuir-Blodgett Deposited Ordered Graphene Assemblies for Dengue Detection.

In this manuscript partially reduced graphene oxide (RGO) nanosheet-based electrodes have been utilized for quantification of the NS1 protein and subsequently for dengue detection. NS1 is the biomarker found circulating in the body of dengue-infected persons on or after first day of the appearance of disease symptoms. Graphene oxide (GO) has been synthesized using a modified Hummer's method, and its ordered nanostructured films have been electrophoretically deposited on indium tin oxide (ITO)-coated glass substrates using Langmuir-Blodgett (LB) deposition. Deposited LB films of GO have been reduced with hydrazine vapors to obtain RGO-coated ITO electrodes. NS1 antibodies have been grafted onto the ordered thin films using covalent linking, and the bioelectrodes have been utilized for the specific detection of NS1 antigen. The electrochemical performance of the fabricated bioelectrodes for NS1 antigen detection has been explored in standard and spiked sera samples. The limit of detection for the standard samples and spiked serum samples is found to be 0.069 ng mL-1 and 0.081 ng mL-1, respectively, with a sensitivity of 8.41 and 36.75 Ω per ng mL, respectively, in the detection range of 101 to 107 ng mL-1.

Lipid-lipid interactions in aminated reduced graphene oxide interface for biosensing application.

A label-free biosensor based on antiapolipoprotein B 100 functionalized-aminated reduced graphene oxide interface has been fabricated for detection of low density lipoprotein (LDL or lipid) cholesterol. The aminated reduced graphene oxide (NH2-rGO) based electrode surface is covalently functionalized with antiapolipoprotein B 100 (AAB or lipid) using EDC/NHS coupling chemistry. The lipid-lipid interactions at the NH2-rGO electrode surface have been investigated using electrochemical impedance spectroscopic technique. The structural and morphological investigations of NH2-rGO based immunosensor have been accomplished via transmission electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, UV-visible, and electrochemical techniques. The impedimetric response of the proposed immunosensor shows excellent sensitivity (612 Ω mg(-1) dL cm(-2)), a response time of 250 s, and a low detection limit of 5 mg/dL of LDL molecules. The association, dissociation, and equilibrium rate constants for this immunoelectrode are found to be 1.66 M(-1) s(-1), 0.6 s(-1), and 2.77 M(-1), respectively. The long-term stability and excellent reproducibility of the proposed immunosensor indicates a suitable platform for detection of LDL or lipid molecules. This immunosensor provides an efficient platform for analysis of the antigen-antibody interactions of lipid molecules.

A novel approach to low-cost, rapid and simultaneous colorimetric detection of multiple analytes using 3D printed microfluidic channels.

This research paper presents an inventive technique to swiftly create microfluidic channels on distinct membrane papers, enabling colorimetric drug detection. Using a modified DIY RepRap 3D printer with a syringe pump, microfluidic channels (µPADs) are crafted on a flexible nylon-based substrate. This allows simultaneous detection of four common drugs with a single reagent. An optimized blend of polydimethylsiloxane (PDMS) dissolved in hexane is used to create hydrophobic channels on various filter papers. The PDMS-hexane mixture infiltrates the paper's pores, forming hydrophobic barriers that confine liquids within the channels. These barriers are cured on the printer's hot plate, controlling channel width and preventing spreading. Capillary action drives fluid along these paths without spreading. This novel approach provides a versatile solution for rapid microfluidic channel creation on membrane papers. The DIY RepRap 3D printer integration offers precise control and faster curing. The PDMS-hexane solution accurately forms hydrophobic barriers, containing liquids within desired channels. The resulting microfluidic system holds potential for portable, cost-effective drug detection and various sensing applications.

Tin oxide quantum dot based DNA sensor for pathogen detection.

We report the application of nano crystalline tin oxide quantum dots (SnO2-QDs) for electrochemical detection of Vibrio cholerae based on DNA hybridization technique. SnO2-QDs (- 1-5 nm) have been synthesized by laser ablation technique in liquid (LAL) and electrophoretically deposited onto hydrolyzed surface of indium tin oxide (ITO) coated glass electrode. A single stranded oligonucleotide probe (23 bases) have been designed form the virulent gene sequence of V. cholerae and has been immobilized onto SnO2-QDs/ITO surface for the fabrication of ssDNA/SnO2-QDs/ITO bioelectrode and these bioelectrode have been further used for DNA hybridization (dsDNA/SnO2-QDs/ITO). The electrochemical response studies have been carried out with different concentration genomic DNA (100-500 ng/microL), which indicated that SnO2 provides an effective surface to bind with the phosphate group of DNA, thus resulting in an enhanced electron transport. The hybridized electrode exhibits linear response with regression coefficient (R) 0.974, high sensitivity 35.20 nA/ng/cm2, low detection limit (31.5 ng/microL), faster response time (3 s) and high stability of 0-120 days when stored under refrigerated conditions.

Surfactant-promoted formation of fractal and dendritic nanostructures of gold and silver at the organic-aqueous interface.

The effect of surfactants such as tetraoctylammoniumbromide (TOAB) and cetyltrimethylammoniumbromide (CTAB) on the type of nanostructures formed when gold ions present in the organic phase are reduced at the interface by hydrazine in the aqueous phase has been investigated. Extended fractal structures are formed at the liquid-liquid interface, the fractal structures themselves comprising cauliflower type units formed by gold nanorods. Accordingly, the nanostructures exhibit transverse and longitudinal plasmon adsorption bands in the 550 and 800 nm regions, respectively. Dendritic structures of silver are formed at the interface when Ag ions are reduced similarly in the presence of surfactants. The nanostructures consist of nanoparticles or nanorods with five-fold symmetry.

Nature and properties of ultrathin nanocrystalline gold films formed at the organic-aqueous interface.

Ultrathin nanocrystalline films of gold formed at different temperatures at the organic-aqueous interface have been investigated by X-ray diffraction, electron microscopy, atomic force microscopy, and electronic spectroscopy. The films are smooth and continuous over relatively large length scales and are generally approximately 100 nm thick. The size of the nanocrystals is sensitive to the reaction temperature, which also determines whether the film is metallic or an activated conductor. The surface plasmon band of gold is highly red-shifted in the films. Alkanethiols perturb the structure of the films, with the magnitude of the effect depending on the chain length. Accordingly, the position of the plasmon band and the electrical resistance of the films are affected by interaction with alkanethiols; the plasmon band approaches that of isolated nanocrystals in the presence of long-chain thiols.

Use of the liquid-liquid interface for generating ultrathin nanocrystalline films of metals, chalcogenides, and oxides.

The air-water interface has traditionally been employed to prepare particle assemblies and films of metals and semiconductors. The interface between water and an organic liquid, however, has not been investigated sufficiently for possible use in preparing nanocrystals and thin films of materials. In this article, we demonstrate the use of the liquid-liquid interface as a medium for preparing ultrathin films of metals, chalcogenides and oxides. The method involves the reaction at the interface between a metal-organic compound in the organic layer and an appropriate reagent for reduction, sulfidation, etc. in the aqueous layer. Some of the materials discussed are nanocrystalline films of gold, CuS, CuSe, CuO, and Cu(OH)2 formed at the liquid-liquid interface. The results reported in this article should demonstrate the versatility and potential of the liquid-liquid interface for preparing nanomaterials and ultrathin films and encourage further research in this area.

Self assembled DC sputtered nanostructured rutile TiO2 platform for bisphenol A detection.

A novel biosensor platform comprising of the functionalized sputtered rutile nanostructured titanium dioxide (nTiO2) for rapid detection of estrogenic substance (bisphenol A) has been proposed. The direct current (DC) sputtering of titanium (Ti) on glass substrate has been converted to ordered nanostructured TiO2 film via oxidation. The nanostructured TiO2 surface was functionalized with self-assembled monolayer (SAM) of 3-aminopropyltriethoxysilane (APTES) and glutaraldehyde. The enzyme molecule, tyrosinase (Tyrs) has been covalently immobilized on the surface of APTES modified nanostructured TiO2 film. To investigate the crystalline structure and surface morphology of functionalized nTiO2/Ti electrode, the X-ray diffraction, scanning electron microscopy, atomic force microscopy and Fourier transform infrared spectroscopy have been carried out. This impedimetric biosensor exhibits a comparable sensitivity (361.9 kΩ/µM) in a wide range of detection (0.01-1.0 µM) and a response time of 250 s for bisphenol A (BPA) monitoring. This novel manufacturing process for nTiO2 film is cheap, practical and safer for functionalization with SAM and glutaraldehyde to improve the biosensor efficacy. The strong protein absorption capability of the nTiO2 surface demonstrates an excellent electrochemical biosensor and could be useful for the detection of other phenolic compounds.

Tyrosinase conjugated reduced graphene oxide based biointerface for bisphenol A sensor.

We have fabricated a nanocomposite of reduced graphene oxide (rGO) sheets and chitosan (Cn) polymer based highly sensitive electrochemical biosensor for detection of bisphenol A (BPA). The two-dimensional structure and chemical functionality of rGO and Cn provide an excellent electrode surface for loading of tyrosinase enzyme molecules. This rGO-Cn nanocomposite is capable of effectively utilizing their superior conductivity, larger effective surface area and superior electrochemical performance due to its synergistic effect between rGO and Cn. The structural, morphological and electrochemical characterizations of nanocomposite sheets have been performed by electron microscopy, X-ray diffraction, FTIR and Potentiostat/Galvanostat techniques. This fabricated biosensor is sensitive to nanomolar (0.74 nM) concentration of BPA and detection time is 10s compared to conventional BPA ELISA kit (0.3 µg/L and 2.5h). The rGO-Cn based biosensor exhibits a higher sensitivity (83.3 µA nM(-1) cm(-2)), wider linearity (0.01-50 µM) with good selectivity towards BPA. This biosensor is capable to quantify real sample of BPA using packaged drinking water bottles. This rGO-Cn nanocomposite sheets emerges as a potential electrode material for detection of other estrogenic substrate.

A Label-Free Photoluminescence Genosensor Using Nanostructured Magnesium Oxide for Cholera Detection.

Nanomaterial-based photoluminescence (PL) diagnostic devices offer fast and highly sensitive detection of pesticides, DNA, and toxic agents. Here we report a label-free PL genosensor for sensitive detection of Vibrio cholerae that is based on a DNA hybridization strategy utilizing nanostructured magnesium oxide (nMgO; size >30 nm) particles. The morphology and size of the synthesized nMgO were determined by transmission electron microscopic (TEM) studies. The probe DNA (pDNA) was conjugated with nMgO and characterized by X-ray photoelectron and Fourier transform infrared spectroscopic techniques. The target complementary genomic DNA (cDNA) isolated from clinical samples of V. cholerae was subjected to DNA hybridization studies using the pDNA-nMgO complex and detection of the cDNA was accomplished by measuring changes in PL intensity. The PL peak intensity measured at 700 nm (red emission) increases with the increase in cDNA concentration. A linear range of response in the developed PL genosensor was observed from 100 to 500 ng/µL with a sensitivity of 1.306 emi/ng, detection limit of 3.133 ng/µL and a regression coefficient (R(2)) of 0.987. These results show that this ultrasensitive PL genosensor has the potential for applications in the clinical diagnosis of cholera.

Biological delignification of paddy straw and Parthenium sp. using a novel micromycete Myrothecium roridum LG7 for enhanced saccharification.

A new lignolytic micromycete fungus Myrothecium roridum LG7 was isolated and selected for biological delignification of agro residue-paddy straw and herbaceous weed Parthenium sp. Physical and chemical modifications in the biomass following pretreatment with M. roridum LG7 for 7 days in term of structural modification and lignin removal, changes in lignin skeleton, and alteration of cellulose crystallinity was observed through SEM-EDXA, FTIR and XRD analysis, respectively. Colonization of the fungus led to high amount of lignin removal (5.8-6.98mg/gds) from pretreated biomass which could be recovered as a value added product. Enzymatic hydrolysis of M. roridum LG7 pretreated biomass released significantly higher amount of reducing sugars (455.81-509.65 mg/gds) as compared to respective raw biomass within 24h. This study illustrates the promise of M. roridum LG7 for biological pretreatment through structural and chemical alteration of biomass beside creation of alkaline environment which prevent the growth of other contaminants.

A highly efficient microfluidic nano biochip based on nanostructured nickel oxide.

We present results of the studies relating to fabrication of a microfluidic biosensor chip based on nickel oxide nanorods (NRs-NiO) that is capable of directly measuring the concentration of total cholesterol in human blood through electrochemical detection. Using this chip we demonstrate, with high reliability and in a time efficient manner, the detection of cholesterol present in buffer solutions at clinically relevant concentrations. The microfluidic channel has been fabricated onto a nickel oxide nanorod-based electrode co-immobilized with cholesterol esterase (ChEt) and cholesterol oxidase (ChOx) that serves as the working electrode. Bare indium tin oxide served as the counter electrode. A Ag/AgCl wire introduced to the outlet of the microchannel acts as a reference electrode. The fabricated NiO nanorod-based electrode has been characterized using X-ray diffraction, Raman spectroscopy, HR-TEM, FT-IR, UV-visible spectroscopy and electrochemical techniques. The presented NRs-NiO based microfluidic sensor exhibits linearity in the range of 1.5-10.3 mM, a high sensitivity of 0.12 mA mM(-1) cm(-2) and a low value of 0.16 mM of the Michaelis-Menten constant (Km).

Microfluidic-integrated biosensors: prospects for point-of-care diagnostics.

There is a growing demand to integrate biosensors with microfluidics to provide miniaturized platforms with many favorable properties, such as reduced sample volume, decreased processing time, low cost analysis and low reagent consumption. These microfluidics-integrated biosensors would also have numerous advantages such as laminar flow, minimal handling of hazardous materials, multiple sample detection in parallel, portability and versatility in design. Microfluidics involves the science and technology of manipulation of fluids at the micro- to nano-liter level. It is predicted that combining biosensors with microfluidic chips will yield enhanced analytical capability, and widen the possibilities for applications in clinical diagnostics. The recent developments in microfluidics have helped researchers working in industries and educational institutes to adopt some of these platforms for point-of-care (POC) diagnostics. This review focuses on the latest advancements in the fields of microfluidic biosensing technologies, and on the challenges and possible solutions for translation of this technology for POC diagnostic applications. We also discuss the fabrication techniques required for developing microfluidic-integrated biosensors, recently reported biomarkers, and the prospects of POC diagnostics in the medical industry.

Magnesium oxide grafted carbon nanotubes based impedimetric genosensor for biomedical application.

Nanostructured magnesium oxide (size<10nm) grafted carboxyl (COOH) functionalized multi-walled carbon nanotubes (nMgO-cMWCNTs) deposited electrophoretically onto indium tin oxide (ITO) coated glass electrode and have been utilized for Vibrio cholerae detection. Aminated 23 bases single stranded DNA (NH2-ssDNA) probe sequence (O1 gene) of V. cholerae has been covalently functionalized onto nMgO-cMWCNTs/ITO electrode surface using EDC-NHS chemistry. This DNA functionalized MgO grafted cMWCNTs electrode has been characterized using X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), transmission electron microscopy (TEM) and electrochemical techniques. The results of XPS studies reveal that sufficient O-C=O groups present at the nMgO-cMWCNTs surface are utilized for DNA binding. The results of hybridization studies conducted with fragmented target DNA (ftDNA) of V. cholerae using electrochemical impedance spectroscopy (EIS) reveal sensitivity as 3.87 Ω ng(-1) cm(-2), detection limit of ~21.70 ng µL(-1) in the linear range of 100-500 ng µL(-1) and stability of about 120 days. The proposed DNA functionalized nMgO-cMWCNTs nanomatrix provides a novel impedimetric platform for the fabrication of a compact genosensor device for biomedical application.

Phase control of nanostructured iron oxide for application to biosensor.

We report results of the studies relating to the phase transformation of bare Fe3O4 nanoparticles (NPs) to α-Fe2O3 NPs obtained during electrophoretic film deposition onto indium-tin oxide coated glass plates. The in situ oxidation of NPs during electrophoretic deposition can be circumvented using surface passivation of the Fe3O4 NPs with an organic shell (carbon) as well as an inorganic shell (silica), while retaining the biocompatibility of the Fe3O4 NPs. XRD and XPS studies reveal the transformation of Fe3O4 NPs to α-Fe2O3 NPs upon electrophoretic deposition, and the retention of the phase of the Fe3O4 NPs upon encapsulation with carbon and silica, respectively. The results of SEM studies indicate decreased agglomeration of the Fe3O4 NPs upon encapsulation during film deposition. Attempts have been made to compare the characteristics of cholesterol biosensors fabricated using Fe3O4@C and α-Fe2O3 NPs, respectively. The Fe3O4@C NPs based cholesterol biosensor shows response time of 60 s, a linearity range of 25-500 mg dl-1, a sensitivity of 193 nA mg-1 dl cm-2 and a Michaelis-Menten constant of 1.44 mg dl-1.

Biocompatible nanostructured magnesium oxide-chitosan platform for genosensing application.

A novel organic-inorganic platform comprising of chitosan (CH) modified nanostructured magnesium oxide (nanoMgO) has been electrophoretically deposited on the indium-tin-oxide (ITO) substrate. The single stranded probe DNA (ssDNA) sequence of Vibrio cholerae has been covalently functionalized onto CH-nanoMgO/ITO surface. The cytotoxicity assay of nanoMgO particles, examined using human intestinal cell line (INT 407), reveals no significant cytotoxicity at the given doses in the range of 50-350 µg/mL. The X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR) and various microscopic techniques have been employed for the structural and morphological analysis of the fabricated electrodes. The electrochemical response studies of ssDNA and fragmented genomic DNA hybridized electrode (dsGDNA/CH-nanoMgO/ITO) have been carried out using cyclic voltammetry (CV) and differential pulse voltammetry (DPV) techniques. The dsGDNA/CH-nanoMgO/ITO bioelectrode exhibits a linear response in the range 100-500 ng/µL with improved sensitivity of 36.72 nA/ng/cm(2), faster response time of 3s and high stability of 3-4 months under refrigerated condition. The lower detection limit of fabricated genosensor has been estimated as 35.20 ng/µL and it shows good reproducibility/repeatability.

Self-assembled monolayer based impedimetric platform for food borne mycotoxin detection.

A self-assembled monolayer (SAM) of 11-amino-1-undecanethiol (AUT) has been fabricated onto a gold (Au) substrate to co-immobilize anti-ochratoxin-A antibodies (AO-IgGs) and bovine serum albumin (BSA) to detect food borne mycotoxin [i.e., ochratoxin-A (OTA)]. AUT/Au electrode, AO-IgGs/AUT/Au immunoelectrode and BSA/IgGs/AUT/Au immunoelectrode have been characterized using scanning electron microscopy (SEM) and electrochemical studies such as cyclic voltammetry (CV), differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS). Electrochemical studies reveal that the AUT-SAM with NH2 groups provide favorable conditions to immobilize AO-IgGs with better orientation, resulting in enhanced electron transport to obtain improved sensing characteristics. The EIS response studies of the BSA/AO-IgGs/AUT/Au immunoelectrode obtained as a function of OTA concentration reveal that the value of the charge transfer resistance (RCT) increases with increased OTA concentration. The BSA/AO-IgGs/AUT/Au immunoelectrode exhibits linearity over 0.5-6.0 ng/dl, detection limit of 0.08 ng/dl using 3σb/m criteria, response time of 30 s and sensitivity of ∼36.83 Ω/ng dl(-1) cm(-2) with a regression coefficient of 0.999. Attempts have been made to monitor the change in RCT of BSA/AO-IgGs/AUT/Au immunoelectrode on addition of coffee samples.

Effects of changes in the interparticle separation induced by alkanethiols on the surface plasmon band and other properties of nanocrystalline gold films.

Effects of changing the interparticle separation on the surface plasmon bands of ultrathin films of gold nanoparticles have been investigated by examining the interaction of alkanethiols of varying chain length on nanocrystalline gold films generated at the organic-aqueous interface. Adsorption of alkanethiols causes blue-shifts of the surface plasmon adsorption band, the magnitude of the shift being proportional to the chain length. The disordered nanocrystals thus created (lambdamax, 530 m) are in equilibrium with the ordered nanocrystals in the film (lambdamax, 700 m) as indicated by an isosbestic point around 600 nm. Long chain thiols disintegrate or disorder the gold films more effectively, as demonstrated by the increased population of the thiol-capped gold nanocrystals in solution. The rate of interaction of the thiols with the film decreases with the decreasing chain length. The effect of an alkanethiol on the spectrum of the gold film is specific, in that the effects with long and short chains are reversible. The changes in the plasmon band of gold due to interparticle separation can be satisfactorily modeled on the basis of the Maxwell-Garnett formalism. Spectroscopic studies, augmented by calorimetric measurements, suggest that the interaction of alkanethiols involves two steps, the first step being the exothermic gold film-thiol interaction and the second step includes the endothermic disordering process followed by further thiol capping of isolated gold particles.

Nanocrystalline films of Au-Ag, Au-Cu, and Au-Ag-Cu alloys formed at the organic-aqueous interface.

By the reaction of the appropriate organometallic precursors and tetrakishydroxymethyl phosphonium chloride (THPC) at the toluene-water interface, we have prepared nanocrystalline films of Au-Ag and Au-Cu alloys with a range of compositions. The films have been characterized by X-ray diffraction, electron microscopy, optical spectroscopy, and other techniques, besides compositional analysis. The particle size of the nanocrystals in the films varies with the composition. The surface plasmon band of the alloy films as well as of the organosols obtained by the disintegration of the films by the addition of an alkanethiol show the expected composition dependence. It has been possible to prepare the nanocrystalline films of a ternary Au-Ag-Cu alloy.